Pharm lyse solution

Circulating EPCs were detected by flow cytometry as described previously^{20 (link)}. Anticoagulated peripheral blood (200 μL) was incubated for 30 min in the dark with a fluorescein isothiocyanate (FITC)-conjugated human CD34 antibody (Clone 581; BD Biosciences) and a PE-conjugated human KDR antibody (#560872, BD Biosciences). To assess the background, isotype controls were used as negative controls based on the species and immunoglobulin G subclass of each antibody. Erythrocytes were lysed with 2 mL of PharM Lyse solution (BD Pharmingen) for 7 min at room temperature, and the remaining cells were washed with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin. The cells were suspended in 400 μL of 2% paraformaldehyde and analyzed on a flow cytometer. The number of circulating EPCs was determined by the ratio of CD34⁺KDR⁺ cells per 100 PBMNCs.

For each sample, 20 × 10⁶ leukocytes were processed as already described^{7 (link)} within 4 hours from material collection. In details, the sample volume containing 20 × 10⁶ leukocytes underwent an erythrocyte-lysis step, with 45 mL of Pharm Lyse solution (BD Biosciences) for 15 minutes at RT, under gentle agitation. Samples were then centrifuged (400 g, 10 min, room temperature) and washed by adding 2 mL of Stain Buffer (BD Biosciences). The pellet of each sample was added to the lyophilized cocktail of reagents, previously re-hydrated by the addition of 100 µl of Stain Buffer (BD Biosciences); 1 µM Syto16 (Thermo Fisher Scientific, Eisai, Medipost - US) was finally added, as liquid drop-in, to each tube. Samples were incubated in the dark for 30 minutes at 4 °C, washed with 2 mL of Stain Buffer (BD Biosciences), centrifuged (400 g, 10 min, room temperature), and re-suspended in 1.5 mL of FACSFlow (BD Biosciences).

Femurs from 7 weeks old WT mice were collected and rapidly flushed in sterile conditions with RPMI/10% FBS and smashed through a 100 μm filter to remove aggregates and centrifuge at 1,400 rpm for 5 min. The pellet was resuspended in PharmLyse solution (BD Biosciences) to lyse red blood cells. Lysis reaction was stopped by adding FBS and then centrifuged 5 min at 1,400 rpm. Pellet was resuspended in PBS-FBS 0.5%-EDTA 2 mM buffer prior to isolation. Neutrophils were isolated using the Neutrophil Isolation Kit (MiltenyiBiotec) following manufacturer instructions. Briefly, cells were stained with a cocktail of biotin-conjugated monoclonal antibodies not expressed on neutrophils. After that, cells were stained with magnetic anti-biotin antibodies and separated using magnetic LS Columns (MiltenyiBiotec). Cells unretained by the colums were numerated and seeded in a 96 well-plate at 1.10⁵ cells per well prior to stimulation. Supernatant was collected after 4 h stimulation and stored at −80°C for further analysis. Cell death was monitored by MTT using a standard protocol. Thiazollyl blue tetrazolium bromide (Sigma) solution was added onto the cells after supernatant collection and incubated for 2 h at 37°C, and a 10% SDS acetic acid solution was then added. MTT reduction to formazan was quantified by an absorbance microplate reader (EL800, BioTek, Colmar) at 610 nm (KC4 software).

PBMCs were isolated from fresh whole blood (AllCells) via Leucosep tube (Greiner Bio-One), including red blood cell lysis using Pharm Lyse solution (BD Biosciences). For viability experiments, unstimulated PBMCs were plated at 200,000 cells per well in 100 µl growth media (RPMI 1640 supplemented with 5% FBS, 2 mM l-glutamine, 10 mM HEPES and 100 IU penicillin/100 µg ml⁻¹ streptomycin) in 96-well clear polystyrene round-bottom tissue culture-treated plates. A total of 50 µl of growth media and 50 µl of 4× compound stock solution (7-point log dilutions, final concentration range of 0.025–25,000 nM, 0.25% DMSO) were immediately added, and the cells were cultured at 37 °C with 5% CO₂ for 24 h. Subsequently, plates were centrifuged at ~500g for 5 min at room temperature, and 100 µl of supernatant was removed for subsequent cytokine analysis. For viability determination, 100 µl of CTG (Promega) was added to the remaining 100 µl cells or growth media. Plates were shaken for 2 min, and the reaction was transferred to a black-wall, clear flat-bottom 96-well polystyrene plate for assay readout. The assay plate was allowed to sit statically for 10 min at room temperature before measuring luminescence using an M1000 Pro plate reader (Tecan).

For each sample, 20 x 10⁶ leukocytes were processed within 4 h from blood collection, as described [32 (link)]. Briefly, the sample volume containing 20 x 10⁶ leukocytes underwent an erythrocyte lysis step through the addition of 45 mL of Pharm Lyse solution (BD) for 15 min at room temperature under gentle agitation, as per the manufacturer’s instructions. Samples were then centrifuged (400 x g for 10 min at room temperature) and washed by adding 2 mL of staining buffer, containing bovine serum albumin (BD). Surface staining was carried out by adding the pellet of each sample to the re-hydrated lyophilized cocktail of reagents. To this, 1 μM Syto-16 (Thermo Fisher Scientific, Waltham, MA, USA) and V450-conjugated anti-CD144 (i.e. conjugated with fluorochrome V450, with 406 nm excitation and 450 nm emission) were added as a liquid drop to each panel tube (Fgures 3 and 4). Samples were incubated in the dark for 30 min at 4°C, washed into 2 mL of staining buffer with bovine serum albumin, and resuspended in 1.5 mL of FACSFlow buffer (BD).